PROJECT SUMMARY Antimicrobial resistance (AMR) is an increasingly prevalent and serious problem worldwide. Most often, AMR arises from horizontal gene transfer (HGT), involving mobile genetic elements (MGE) such as plasmids and phages. The human gut is a hotspot for both the evolution and spread of AMR; commensals serve as a source of AMR via HGT. Slowing the evolution and spread of AMR is both possible and necessary. Knowledge about the evolutionary history of AMR development and dissemination in vivo is essential to facilitate effective stewardship. Yet, this knowledge remains limited. Important aspects of AMR evolution become evident only in complex environments and in the setting of diverse communities. We will study responses of the human gut microbiota to antibiotic exposure in vivo, as well as of complex stool-derived subject-specific synthetic communities in vitro, at high temporal resolution and using innovative approaches. We will link AMR and other MGE-associated genes to their host core genomes using high resolution chromosome conformation capture (Hi-C) and monitor these genes and elements in bacterial hosts before, during and after antibiotic exposure. The short term-objectives of the proposed work are to assess the types and rates of de novo mutation and HGT involving AMR genes that occur in the human gut microbiota during antibiotic exposure. The long- term objectives are to improve antibiotic stewardship by identifying critical events or transitions in the evolution and dissemination of AMR in vivo, and the factors and conditions that make those events less likely. Aim 1. Characterize the associations among antimicrobial resistance genes, mobile genetic elements, and microbial strains in the gut of healthy humans. We will use Hi-C and metagenomic sequencing to recover microbial population genomes from stool samples of 20 healthy adults and track their abundance over an antibiotic-free interval of 5 months. Aim 2. Describe how ciprofloxacin exposure affects the associations among antimicrobial resistance genes, mobile genetic elements, and microbial strains in healthy humans. We will apply the same methods to additional stool samples collected from the same 20 healthy adults after Cp exposure. Host ranges of MGE and AMR genes, and other genomic features will be compared before, during and after Cp exposure. Aim 3. Assess the effects of ciprofloxacin in vitro on the mobilization of antimicrobial resistance genes and transfer of mobile genetic elements in a synthetic, human gut-derived microbial community. After isolating ~100 diverse microbial strains from a pre-treatment stool sample of each of the 20 healthy adults, they will be sequenced, pooled, and then exposed to ciprofloxacin in vitro. Each strain will then be re- sequenced to identify de novo mutations. To identify MGE, communities will be characterized with Hi-C before, during and after exposure.